JP2024112294A - vaccine - Google Patents

Abstract

To provide an animal vaccine.SOLUTION: A specific cationic lipid is mixed with an antigen to produce a vaccine.SELECTED DRAWING: None

Description

The present invention relates to a vaccine for animals.

Cationic lipids have been reported as carriers for delivering nucleic acids and the like into cells (Patent Documents 1 to 3). Specifically, the cationic lipid used in the examples of Patent Document 1 is COATSOME (registered trademark) SS-E, the example of Patent Document 2 is COATSOME (registered trademark) SS-EC, and the example of Patent Document 3 is COATSOME (registered trademark) SS-OP.

Non-Patent Document 1 discloses COATSOME (registered trademark) SS-OP and COATSOME (registered trademark) SS-EC as cationic lipids. Non-Patent Document 1 reports that COATSOME (registered trademark) SS-EC may be applicable as an adjuvant or DNA vaccine. However, the adjuvant function of COATSOME (registered trademark) SS-EC when actually combined with an antigen has not been confirmed.
In addition, it has been reported that lipid nanoparticles (LNPs) have an adjuvant effect (Non-Patent Document 2).

The adjuvant activity of cationic lipids has started to attract attention since a report was released that liposomes containing cationic lipids have adjuvant activity. The cationic lipids used in the report are cationic lipids that have a wide positive charge in the neutral to acidic range, and are highly toxic compared to their adjuvant activity, so they have not been put to practical use. The mechanism of the adjuvant effect is also unclear. On the other hand, the cationic lipids contained in LNPs used in mRNA vaccines are characterized by being positively charged only in the acidic range, and it is speculated that the adjuvant activity is also different because the action points on cells are clearly different (Non-Patent Document 3).
The adjuvant effect of mRNA-LNP was reported in 2018, but it was unclear whether the action was due to mRNA or cationic lipid. In Non-Patent Document 4, it was revealed that the use of LNP without mRNA has an adjuvant effect, and it was also shown that LNP without cationic lipid has no adjuvant activity. Non-Patent Document 5 also shows that administration of LNP without mRNA induces an inflammatory reaction (immune response). From these findings, it was proven that the adjuvant activity of mRNA-LNP is due to cationic lipid.
Therefore, cationic lipids are expected to be used as adjuvants.

Adjuvants can be substances that, when used in combination with an antigen, enhance the immunogenicity of the antigen, accelerate the immune response to the antigen, extend the immune response to the antigen, and/or switch to a different immune response (e.g., switch from Th1 immune response to Th2 immune response or switch from humoral immunity to cellular immunity) than the immune response induced by the antigen alone. Thus, adjuvants are useful for reducing the dose or number of administrations of a vaccine, or the required amount of antigen in a vaccine. Furthermore, adjuvants are essential components in the veterinary industry for mixing and simultaneously immunizing multiple types of antigens. Adjuvants currently mainly used in Japan include mineral acid salts (e.g., adjuvants whose main component is an aluminum salt, such as aluminum hydroxide or aluminum phosphate (also called "alum adjuvant"). Alum adjuvants are the oldest and most widely used adjuvants, but because they are not soluble, they are difficult to mix uniformly with antigens, and although they induce humoral immunity, they do not induce cellular immunity very well. In addition, they have problems with side reactions such as fever and induction of allergic reactions (IgE). In addition, oil adjuvants are used exclusively for veterinary purposes. Oil adjuvants have a strong adjuvant effect, but the problem is that they cause strong side effects in animals to which they are administered (e.g., local swelling, systemic symptoms such as fever, and weight loss). For this reason, the animal medical industry has been eager to develop an adjuvant that has fewer side effects and is more effective than alum adjuvants and oil adjuvants.

Patent No. 6093710 Patent No. 6875685 WO2019/188867

Hidetoshi Akita, Pharmacology, 82(2), 79-83(2022) Joanna L. Turley and Ed C. Lavelle, Immunity 54 2695-2697, December 14, 2021 R, Verbeke et al., Immunity 55, 1993-2005, November 8, 2022 Mohamad-Gabriel Alameh et. al., Immunity 54 2877-2892, December 14, 2021 S. Ndeupen et al., iScience 24, 103479, December 17, 2021

The objective of the present invention is to provide a vaccine containing an adjuvant that has excellent immunostimulatory activity and few side effects.

The present inventors have discovered that the cationic lipid represented by the general formula (I) described below functions successfully as an adjuvant, and have completed the present invention.

That is, the present invention can be exemplified as follows.
[1]
A vaccine comprising a cationic lipid and an antigen,
A vaccine, wherein the cationic lipid is a compound represented by general formula (I) described below.
[Wherein,
Each of na and nb independently represents an integer of 1 to 4.
^Xa and ^Xb each independently represent the structure ^X1 described below or the structure ^X2 described below;
^R7 represents an alkyl group having 1 to 6 carbon atoms;
p represents an integer of 1 to 4;
R ^1a and R ^1b each independently represent R ³ or R ⁴ ;
^R3 represents -( _CH2 ) _n1- ;
^R4 represents _-C6H4- ( _CH2 ) _n3 - _COO- ( _CH2 ) _n2- ;
n1 represents an integer of 1 to 5;
n2 represents an integer of 1 to 5;
n3 represents an integer from 1 to 3;
^R2a and ^R2b each independently represent ^R5 or ^R6 ;
^R5 represents a functional group obtained by removing a carboxy group from a monoester of succinic acid or glutaric acid with a fat-soluble vitamin having a hydroxyl group,
^R6 represents an aliphatic hydrocarbon group having 12 to 22 carbon atoms.
[2]
The vaccine according to [1], which satisfies any one of the following conditions (1) to (4) and any one of the following conditions (5) to (8):
(1) na is 2, ^Xa is ^X1 , ^R1a is R3 in which n1 is ³ , and ^R2a is ^R5 ;
(2) na is 2, ^Xa is ^X2 , ^R1a is ^R3 with n1 being 2, and ^R2a is ^R5 ;
(3) na is 2, ^Xa is ^X2 , ^R1a is ^R3 with n1 being 2, and ^R2a is ^R6 ;
(4) na is 2, ^Xa is ^X2 , ^R1a is ^R4 , n2 is 2 and n3 is 1, and ^R2a is ^R6 ;
(5) nb is 2, ^Xb is ^X1 , ^R1b is R3 with n1 being ³ , and ^R2b is ^R5 ;
(6) nb is 2, ^Xb is ^X2 , ^R1b is ^R3 with n1 being 2, and ^R2b is ^R5 ;
(7) nb is 2, ^Xb is ^X2 , ^R1b is ^R3 with n1 being 2, and ^R2b is ^R6 ;
(8) nb is 2, ^Xb is ^X2 , ^R1b is ^R4 , n2 is 2 and n3 is 1, and ^R2b is ^R6 .
[3]
The vaccine of [1] or [2], wherein p is 3 or 4.
[4]
The vaccine according to any one of [1] to [3], wherein na is the same as nb, ^Xa is the same as ^Xb , ^R1a is the same as ^R1b , and ^R2a is the same as ^R2b .
[5]
The vaccine according to any one of [1] to [4], wherein the cationic lipid is a component of ssPalmEC and/or ssPalmE.
[6]
The vaccine according to any one of [1] to [5], wherein the cationic lipid is a constituent lipid of a lipid membrane structure.
[7]
The vaccine according to [6], wherein the lipid membrane structure further contains a non-cationic lipid. [8]
The vaccine according to [7], wherein the non-cationic lipid is one or more components selected from the group consisting of phospholipids, PEG lipids, and steroids.
[9]
The vaccine described in [8], wherein the steroid is one or more components selected from the group consisting of cholesterol and its derivatives.
[10]
The vaccine according to any one of [1] to [9], wherein the antigen is one or more components selected from the group consisting of pathogens and components derived therefrom.
[11]
The vaccine according to any one of [1] to [10], wherein the antigen is an inactivated antigen.
[12]
The vaccine according to [11], wherein the inactivated antigen is an inactivated virus.
[13]
The vaccine according to [11], wherein the inactivated antigen is an inactivated bacterium.
[14]
The vaccine according to [11], wherein the inactivated antigen is a toxoid.
[15]
The vaccine according to [12], wherein the virus is bovine viral diarrhea virus.
[16]
The vaccine according to [13], wherein the bacterium is Avibacterium paragallinarum.
[17]
The vaccine according to [14], wherein the toxoid is a toxoid of Clostridium septicum.
[18]
A vaccine according to any one of [1] to [17] for preventing, alleviating, or treating an infectious disease in an animal.
[19]
The vaccine according to [18], wherein the animal is a livestock animal or a pet animal.
[20]
The vaccine according to any one of [1] to [19], which is administered by a route selected from the group consisting of intramuscular administration, intradermal administration, subcutaneous administration, and intraocular administration.
[21]
The vaccine according to any one of [1] to [19], which is administered intramuscularly.
[22]
A method for producing a vaccine, comprising the steps of:
The vaccine is a vaccine according to any one of [1] to [21],
mixing said cationic lipid and said antigen.
[23]
The method according to [22], wherein the vaccine is a vaccine with improved functionality.

The present invention provides a vaccine containing an adjuvant that has excellent immunostimulatory activity and few side effects. In one aspect, the vaccine can be used to prevent, reduce, or treat infectious diseases in animals.

FIG. 1 shows the results of neutralizing antibody titer measurement after administration of a vaccine containing ssPalmEC-LNP. FIG. 13 is a graph showing the reaction inhibition rate in chickens administered a vaccine containing ssPalmEC-LNP.

<1> Vaccine The vaccine described in this specification is a vaccine containing a cationic lipid and an antigen.

The cationic lipid contained in the vaccine described herein (hereinafter also simply referred to as "cationic lipid") is a compound represented by the following general formula (I). "Cationic lipid" may mean a lipid that has no net charge at neutral pH but has a net positive charge at acidic pH. The cationic nature of the cationic lipid may be due, for example, to the tertiary amine contained in the cationic lipid. As the cationic lipid, one type of cationic lipid may be used, or two or more types of cationic lipids may be used in combination.

In formula (I), na and nb each independently represent an integer from 1 to 4 (i.e., 1, 2, 3, or 4).

In formula (I), ^Xa and ^Xb each independently represent the following structure ^X1 or the following structure ^X2 :
^R7 represents an alkyl group having 1 to 6 carbon atoms;
p represents an integer from 1 to 4 (ie, 1, 2, 3, or 4).

In formula (I), R ^1a and R ^1b each independently represent R ³ or R ⁴ ;
^R3 represents -( _CH2 ) _n1- ;
^R4 represents _-C6H4- ( _CH2 ) _n3 - _COO- ( _CH2 ) _n2- ;
n1 represents an integer from 1 to 5 (i.e., 1, 2, 3, 4, or 5);
n2 represents an integer from 1 to 5 (i.e., 1, 2, 3, 4, or 5);
n3 represents an integer from 1 to 3 (i.e., 1, 2, or 3).

In formula (I), R ^2a and R ^2b each independently represent R ⁵ or R ⁶ ;
^R5 represents a functional group obtained by removing a carboxy group from a monoester of succinic acid or glutaric acid with a fat-soluble vitamin having a hydroxyl group,
^R6 represents an aliphatic hydrocarbon group having 12 to 22 carbon atoms.

na may or may not be the same as nb. na may in particular be the same as nb. na and nb may each in particular be 2.

^Xa may or may not be identical to ^Xb . ^Xa may in particular be identical to ^Xb .

R ^1a may or may not be the same as R ^1b . R ^1a may in particular be the same as R ^1b .

R ^2a may or may not be the same as R ^2b . R ^2a may in particular be the same as R ^2b .

For example, when R ^1a is R ³ , R ^2a may be R ^5. For example, when R ^1b is R ³ , R ^2b may be R ^5. For example, when R 1a is R ³ , R ^2a may be R ^6. For example, when R ^1b is R ³ , R ^2b may be R ^6. For example, when R ^1a is ^{R 4} , R ^2a may be R ^6. For example, when R ^1b is R ⁴ , R ^2b may be R ⁶ .

n1 may in particular be 2 or 3.

For example, when ^Xa is ^X1 , ^R1a may be R3 with n1 being 3. For example, when ^Xb is ^X1 , ^R1b may be R3 with ⁿ¹ being ^3. For example, when ^Xa is ^X2 , ^R1a may be ^R3 with n1 being 2. For example, when ^Xb is ^X2 , ^R1b may be ^R3 with n1 being 2.

n2 may in particular be 2.

n3 may in particular be 1.

For example, when ^Xa is ^X2 , ^R1a may be ^R4 in which n2 is 2 and n3 is 1. For example, when ^Xb is ^X2 , ^R1b may be ^R4 in which n2 is 2 and n3 is 1.

Regarding ^R4 , ^R4 may also be expressed as " _{* -C6H4-} ( _CH2 ) _n3 -COO-( _CH2 ) _n2 -**", where * is the bonding position to ^R2a- COO or ^R2b -COO, and _{** is the bonding position to Xa or Xb. Regarding R4, -C6H4- may be 1,2-phenylene, 1,3-phenylene, or 1,4-phenylene. Regarding R4, -C6H4-} ^{may in particular} _be ^1,4 _{- phenylene} .

The alkyl group having 1 to 6 carbon atoms may be linear (non-cyclic) or cyclic. The alkyl group having 1 to 6 carbon atoms may be linear or branched. The number of carbon atoms in the alkyl group having 1 to 6 carbon atoms may be preferably 1 to 3. Examples of the alkyl group having 1 to 6 carbon atoms include methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, n-pentyl, isopentyl, neopentyl, tert-pentyl, 1,2-dimethylpropyl, 2-methylbutyl, 2-methylpentyl, 3-methylpentyl, 2,2-dimethylbutyl, 2,3-dimethylbutyl, and cyclohexyl. Examples of the alkyl group having 1 to 6 carbon atoms include preferably methyl, ethyl, n-propyl, and isopropyl. Examples of the alkyl group having 1 to 6 carbon atoms include more preferably methyl.

p may in particular be 3 or 4. p may more in particular be 4.

With respect to ^X2 , the nitrogen atom (N) is bonded to ( _CH2 ) _na or ( _CH2 ) _nb , and a carbon atom in the cyclic structure is bonded to ^R1a or ^R1a . Any carbon atom in the cyclic structure may be bonded to ^R1a or ^R1a . For example, when p is 2 or more (e.g., 3 or 4, particularly 4), the carbon atom at the 4th position when the nitrogen atom (N) is the 1st position may be bonded to ^R1a or ^R1a .

The term "monoester of succinic acid or glutaric acid with a fat-soluble vitamin having a hydroxyl group" (hereinafter simply referred to as "monoester") means a compound obtained by esterifying one of the carboxy groups of succinic acid or glutaric acid with a fat-soluble vitamin having a hydroxyl group. When R ^2a or R ^2b is R ⁵ , R ^2a -COO- or R ^2b -COO
- (i.e., R ⁵ -COO-) is an acyloxy group obtained by removing a hydrogen atom from a carboxy group of a monoesterified product. The above explanation of the functional group obtained by removing a carboxy group from a monoesterified product indicates the structure of the functional group, unless otherwise specified, and does not necessarily indicate the method for producing the functional group. That is, the monoesterified product may or may not be one obtained by reacting a fat-soluble vitamin having a hydroxyl group with succinic acid or glutaric acid. The monoesterified product may be, for example, one obtained by reacting a fat-soluble vitamin having a hydroxyl group with succinic anhydride or glutaric anhydride. In addition, the functional group obtained by removing a carboxy group from a monoesterified product or the acyloxy group obtained by removing a hydrogen atom from a carboxy group of a monoesterified product may or may not be one derived from a monoesterified product.

"Vitamin" is not limited to vitamins themselves, but may include provitamins and vitamin derivatives. Examples of fat-soluble vitamins having a hydroxyl group include retinol, ergosterol, 7-dehydrocholesterol, calciferol, corcalciferol, dihydroergocalciferol, dihydrotachysterol, tocopherol, and tocotrienol. A preferred example of a fat-soluble vitamin having a hydroxyl group is tocopherol.

The monoester is preferably a monoester of succinic acid with tocopherol (i.e., a monotocopherol succinate ester) or a monoester of glutaric acid with tocopherol (i.e., a monotocopherol glutarate ester). The monoester is more preferably a monotocopherol succinate ester.

The aliphatic hydrocarbon group having 12 to 22 carbon atoms may be linear (non-cyclic) or cyclic. The aliphatic hydrocarbon group having 12 to 22 carbon atoms may be particularly linear (non-cyclic). The aliphatic hydrocarbon group having 12 to 22 carbon atoms may be linear or branched. The aliphatic hydrocarbon group having 12 to 22 carbon atoms may be saturated or unsaturated. When the aliphatic hydrocarbon group having 12 to 22 carbon atoms is unsaturated, the number of unsaturated bonds contained in the aliphatic hydrocarbon group may be, for example, usually 1 to 6, preferably 1 to 3, and more preferably 1 to 2. The position of the unsaturated bond is not particularly limited. The configuration of the unsaturated bond (i.e., E-Z) is not particularly limited. Examples of the unsaturated bond include a carbon-carbon double bond and a carbon-carbon triple bond. Examples of the preferred unsaturated bond include a carbon-carbon double bond. The number of carbon atoms in the aliphatic hydrocarbon group having 12 to 22 carbon atoms may be preferably 13 to 19, and more preferably 13 to 17. Examples of the aliphatic hydrocarbon group include an alkyl group, an alkenyl group, and an alkynyl group. Examples of the aliphatic hydrocarbon group include an alkyl group and an alkenyl group. Examples of the aliphatic hydrocarbon group having 12 to 22 carbon atoms include a dodecyl group, a tridecyl group, a tetradecyl group, a pentadecyl group, a hexadecyl group, a heptadecyl group, an octadecyl group, a nonadecyl group, an icosyl group, a henicosyl group, a docosyl group, a dodecenyl group, a tridecenyl group, a tetradecenyl group, a pentadecenyl group, a hexadecenyl group, a heptadecenyl group, an octadecenyl group, a nonadecenyl group, an icosenyl group, a henicosyl group, a docosenyl group, a dodecadienyl group, a tridecadien ...decyl group, a decyl group, a decyl group, a decyl group, a decyl group, a decyl group, a decyl group, a decyl group, a decyl group, a decyl group, a decyl group, a decyl group, a decyl group, a decyl group, a decyl group, a decyl group, a decyl group, a decyl group, a decyl group, a decyl group, a decyl group, a Examples of the aliphatic hydrocarbon group having 12 to 22 carbon atoms include a dienyl group, a pentadecadienyl group, a hexadecadienyl group, a heptadecadienyl group, an octadecadienyl group, a nonadecadienyl group, an icosadienyl group, a henicosadienyl group, a docosadienyl group, an octadecatrienyl group, an icosatrienyl group, an icosatetraenyl group, an icosapentaenyl group, a docosahexaenyl group, an isostearyl group, a 1-hexylheptyl group, a 1-hexylnonyl group, a 1-octylnonyl group, a 1-octylundecyl group, and a 1-decylundecyl group. Examples of the aliphatic hydrocarbon group having 12 to 22 carbon atoms include a tridecyl group, a pentadecyl group, a heptadecyl group, a nonadecyl group, a heptadecenyl group, a heptadecadienyl group, and a 1-hexylnonyl group. More preferred examples of the aliphatic hydrocarbon group having 12 to 22 carbon atoms include a tridecyl group, a heptadecyl group, a heptadecenyl group, and a heptadecadienyl group. Particularly preferred examples of the aliphatic hydrocarbon group having 12 to 22 carbon atoms include a heptadecenyl group. The heptadecenyl group may be, for example, an 8-heptadecenyl group. The heptadecenyl group may be, for example, particularly, a (Z)-8-heptadecenyl group.

The aliphatic hydrocarbon group having 12 to 22 carbon atoms may specifically be a functional group obtained by removing a carboxy group from a fatty acid having 13 to 23 carbon atoms. In other words, when R ^2a or R ^2b is R ⁶ , R ^2a -COO- or R ^2b -COO- (i.e., R ⁶ -COO-) may be an acyloxy group obtained by removing a hydrogen atom from the carboxy group of a fatty acid having 13 to 23 carbon atoms. For example, when the fatty acid is linoleic acid, the aliphatic hydrocarbon group having 12 to 22 carbon atoms is an 8,11-heptadecadienyl group (specifically, (8Z,11Z)-8,11-heptadecadienyl group). Also, for example, when the fatty acid is oleic acid, the aliphatic hydrocarbon group having 12 to 22 carbon atoms is an 8-heptadecenyl group (specifically, (Z)-8-heptadecenyl group). Unless otherwise specified, the above explanations regarding the aliphatic hydrocarbon group having 12 to 22 carbon atoms indicate the structure of the functional group, and do not necessarily indicate the method for producing the functional group. That is, for example, a functional group obtained by removing a carboxy group from a fatty acid having 13 to 23 carbon atoms or an acyloxy group obtained by removing a hydrogen atom from the carboxy group of a fatty acid having 13 to 23 carbon atoms may or may not be derived from a fatty acid.

In one embodiment, na may be the same as nb, ^Xa may be the same as ^Xb , ^R1a may be the same as ^R1b , and ^R2a may be the same as ^R2b .

In one embodiment, Xa may be ^X1 , ^R1a may be R3, and ^R2a may be ^R5 . In one embodiment, na may be 2, ^Xa may be ^X1 , R1a may be R3 where n1 is ³ , and ^R2a may be ^R5 . In one embodiment, na may be 2, ^Xa may be ^X1 where R7 is ^methyl , ^R1a may be ^R3 where n1 is ³ , and ^R2a may be a ^residue obtained ^by removing the carboxy group from a monotocopherol succinate ester.

In one embodiment, ^Xb may be ^X1 , ^R1b may be R3, and ^R2b may be ^R5 . In one embodiment, nb may be 2, ^Xb may be ^X1 , R1b may be R3 where n1 is ³ , and ^R2b may be ^R5 . In one embodiment, nb may be 2, ^Xb may be ^X1 where R7 is ^methyl , ^R1b may be ^R3 where n1 is 3, and ^R2b may be a ^residue obtained ^by removing the carboxy group from a monotocopherol succinate ester.

In one embodiment, ^Xa may be ^X2 , ^{R1a may be R3, and R2a may be R5. In one embodiment, na may be 2, Xa may be X2, R1a may be R3 where n1} is 2, and R2a may be ^R5 . In one embodiment, na may be ² , ^Xa may be ^X2 where p is 4, ^R1a may be ^R3 where n1 is 2, ^{and R2a} may be ^a residue obtained by removing the carboxy group from a monotocopherol succinate ester.

In one embodiment, ^Xb may be ^X2 , R ^1b may be ^R3 , and R ^2b may be ^R5 . In one embodiment, nb may be 2, Xb may be ^X2 , R ^1b may be ^R3 where n1 is 2, and R ^2b may be ^R5 . In one embodiment, nb may be 2, ^Xb may be ^X2 where p is 4, R ^1b may be ^R3 where n1 is 2, and ^{R 2b may} be a residue obtained by removing the carboxy group from a monotocopherol succinate ester.

In one embodiment, ^Xa may be X2, ^R1a may be ^R3 and ^R2a may be ^R6 . In one embodiment, na may be ² , ^Xa may be ^X2 , ^R1a may be R3 where n1 is 2 and R2a may be R6. In one embodiment, na may be 2, ^Xa may be ^X2 where p is 4, ^R1a may be ^R3 where n1 is 2 and ^R2a may be an ^{8 -} heptadecenyl group (e.g., ^a (Z)-8-heptadecenyl group).

In one embodiment, X ^b may be X ² , R ^1b may be R ³ and R ^2b may be R ^6. In one embodiment, nb may be 2, X ^b may be X ² , R ^1b may be R ³ where n1 is 2 and R ^2b may be R ^6. In one embodiment, nb may be 2, X ^b may be X ² where p is 4, R ^1b may be R ³ where n1 is 2 and R ^2b may be an 8-heptadecenyl group (e.g., a (Z)-8-heptadecenyl group).

In one embodiment, ^Xa may be ^X2 , ^R1a may be ^R4 , and ^R2a may be ^R6 . In one embodiment, na may be 2, ^Xa may be ^X2 , ^R1a may be ^R4 where n2 is 2 and n3 is 1, and ^R2a may be ^R6 . In one embodiment, na may be 2, ^Xa may be ^X2 where p is 4, ^R1a may be R4 where n2 is 2 and ⁿ³ is 1, and ^R2a may be an 8-heptadecenyl group (e.g., a (Z)-8-heptadecenyl group).

In one embodiment, X ^b may be X ² , R ^1b may be R ⁴ and R ^2b may be R ^6. In one embodiment, nb may be 2, X ^b may be X ² , R ^1b may be R ⁴ where n2 is 2 and n3 is 1 and R ^2b may be R ^6. In one embodiment, nb may be 2, X ^b may be X ² where p is 4, R ^1b may be R 4 where n2 is 2 and ⁿ³ is 1 and R ^2b may be an 8-heptadecenyl group (e.g., a (Z)-8-heptadecenyl group).

Specific examples of cationic lipids include ssPalmE, ssPalmEC, ssPalmOC, and ssPalmOP. Preferred examples of cationic lipids include ssPalmE and ssPalmEC, and more preferred examples include ssPalmEC. ssPalmEC is also referred to as "ssPalmE-P4C2." The structures of ssPalmE, ssPalmEC, ssPalmOC, and ssPalmOP are shown below.

As the cationic lipid, a commercially available product may be used, or one obtained by appropriate production may be used. For example, a commercially available ssPalmE-P4C2 may be COATSOME (registered trademark) SS-EC (manufactured by Yuka Sangyo Co., Ltd.). The method for producing the cationic lipid is not particularly limited. The cationic lipid may be produced, for example, by chemical synthesis. Specifically, the cationic lipid may be produced, for example, by the method described in Japanese Patent No. 6093710, Japanese Patent No. 6875685, or WO2019/188867.

The cationic lipid may function, for example, as an adjuvant. An "adjuvant" may mean a substance that, when used in combination with an antigen, enhances the immunogenicity of the antigen, accelerates the immune response to the antigen, prolongs the immune response to the antigen, and/or switches the immune response to a different immune response than that induced by the antigen alone (e.g., switches from a Th1 immune response to a Th2 immune response or switches from humoral immunity to cellular immunity). An adjuvant may be a single substance or a combination of substances. That is, the cationic lipid may function as an adjuvant alone or in combination with other components.

The cationic lipid may, for example, constitute a lipid membrane structure. In other words, the cationic lipid may, for example, be a constituent lipid of a lipid membrane structure. The cationic lipid may, specifically, be a membrane constituent lipid of a lipid membrane structure. The lipid membrane structure may or may not be made of cationic lipids. That is, the lipid membrane structure may contain other components in addition to the cationic lipid. In other words, the cationic lipid may constitute the lipid membrane structure alone or in combination with other components. In other words, the vaccine described herein may contain other components in addition to the cationic lipid and the antigen. Examples of other components include non-cationic lipids, surfactants, polyethylene glycol (PEG), and proteins. Examples of other components include non-cationic lipids in particular. "Non-cationic lipid" refers to lipids other than cationic lipids. "Non-cationic lipid" may, specifically, refer to lipids that do not have a net positive charge at a selected pH, such as physiological pH. Examples of non-cationic lipids include phospholipids, PEG lipids, glycolipids, peptide lipids, and steroids. Non-cationic lipids include, in particular, phospholipids, PEG lipids, and steroids. Other components include cationic lipids other than the cationic lipid represented by formula (I).

Examples of phospholipids include natural or synthetic phospholipids such as phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), phosphatidylinositol (PI), phosphatidylglycerol (PG), phosphatidic acid (PA), dicetyl phosphate, sphingomyelin (SPM), and cardiolipin; partially or completely hydrogenated versions of these phospholipids; natural lecithins such as soybean lecithin, corn lecithin, cottonseed oil lecithin, and egg yolk lecithin (natural lecithins contain phospholipids); and hydrogenated versions of natural lecithins such as hydrogenated soybean lecithin and hydrogenated egg yolk lecithin. Phospholipids may have acyl groups at the C1 and C2 positions of glycerol. The acyl groups at the C1 and C2 positions can be selected independently. That is, the acyl groups at the C1 and C2 positions may or may not be the same. Examples of the acyl group constituting the phospholipid include acyl groups having 8 to 24 carbon atoms. Examples of the acyl group having 8 to 24 carbon atoms include residues obtained by removing hydroxyl groups from fatty acids such as caprylic acid, pelargonic acid, capric acid, undecanoic acid, lauric acid, tridecanoic acid, myristic acid, pentadecanoic acid, palmitic acid, margaric acid, stearic acid, nonadecanoic acid, arachidic acid, heneicosanoic acid, behenic acid, tricosanoic acid, lignoceric acid, myristoleic acid, palmitoleic acid, oleic acid, eicosenoic acid, erucic acid, hexadecadienoic acid, linoleic acid, eicosadienoic acid, docosadienoic acid, hexadecatrienoic acid, α-linolenic acid, γ-linolenic acid, eicosatrienoic acid, arachidonic acid, eicosapentaenoic acid, docosatetraenoic acid, and docosahexaenoic acid. Examples of the phospholipid include synthetic phospholipids. More preferably, the phospholipid is a synthetic phospholipid containing an unsaturated bond in the acyl group. More preferably, the phospholipid is dioleoylphosphatidylcholine (DOPC) or dioleoylphosphatidylethanolamine (DOPE). Particularly preferably, the phospholipid is dioleoylphosphatidylethanolamine (DOPE).

"PEG lipid" may mean lipids containing modification by PEG. Examples of PEG lipids include PEG phospholipids and diacylglycerol PEG. "PEG phospholipid" may mean phospholipids to which PEG is bound. The phospholipids are as described above. "Diacylglycerol PEG" may mean diacylglycerol to which PEG is bound. Diacylglycerol PEG may have acyl groups at the C1 and C2 positions or the C1 and C3 positions of glycerol. The acyl groups at the C1 and C2 positions or the C1 and C3 positions can be selected independently. That is, the acyl groups at the C1 and C2 positions or the C1 and C3 positions may or may not be the same. Examples of acyl groups constituting diacylglycerol PEG include those exemplified as acyl groups constituting phospholipids. The molecular weight of PEG constituting PEG lipid is not particularly limited. The molecular weight of PEG constituting the PEG lipid may be preferably 200 to 10,000, more preferably 2,000 to 5,000. The PEG lipid preferably includes diacylglycerol PEG in which each acyl group is saturated. The PEG lipid more preferably includes diacylglycerol PEG in which each acyl group is a myristoyl group or a stearoyl group. The PEG lipid further preferably includes diacylglycerol PEG (DMG-PEG2000) in which a myristoyl group is bound to a PEG having a molecular weight of about 2,000, or diacylglycerol PEG (DSG-PEG2000) in which a stearoyl group is bound to a PEG having a molecular weight of about 2,000.
EG2000).

Examples of steroids include sterols and derivatives thereof. Examples of sterols include cholesterol, phytosterol, and dihydrocholesterol. A preferred example of sterol is cholesterol. Examples of sterol derivatives include fatty acid esters of sterol. Examples of sterol derivatives (specifically fatty acid esters) include cholesterol derivatives (specifically fatty acid esters) such as cholesteryl stearate, cholesteryl nonanoate, cholesteryl hydroxystearate, and dihydrocholesteryl oleate. A preferred example of sterol derivatives is cholesteryl stearate. A preferred example of steroids is cholesterol and derivatives thereof. A more preferred example of steroids is cholesterol.

Surfactants include 3-[(3-cholamidopropyl)dimethylammonio]propanesulfonate, sodium cholic acid, octyl glycoside, and N-D-gluco-N-methylalkanamides.

The other ingredients may be commercially available or may be obtained by appropriate manufacturing.

"Lipid membrane structure" may refer to particles having a membrane structure in which the hydrophilic groups of amphipathic lipids are aligned toward the aqueous phase side of the interface. "Amphipathic lipid" may refer to lipids having both hydrophilic and hydrophobic groups. Examples of amphipathic lipids include cationic lipids and phospholipids.

The quantitative ratio of each component constituting the lipid membrane structure is not particularly limited as long as a lipid membrane structure is formed. The content of the cationic lipid in the lipid membrane structure may be, for example, usually 5 to 100 mol%, preferably 5 to 95 mol%, more preferably 10 to 90 mol%, and particularly preferably 20 to 70 mol%. The content of the cationic lipid in the lipid membrane structure may be, for example, usually 5 to 100 mol%, preferably 10 to 90 mol%, and more preferably 20 to 70 mol% relative to the total lipid content in the lipid membrane structure.

The lipid membrane structure can be prepared, for example, by dissolving or dispersing the components of the lipid membrane structure (cationic lipid and optionally other components) in an appropriate solvent or dispersion medium, and carrying out a procedure for inducing organization as necessary. Examples of the solvent or dispersion medium include aqueous solvents and alcoholic solvents. Examples of the procedure for inducing organization include the ethanol dilution method, simple hydration method, ultrasonic treatment method, heating method, vortex method, ether injection method, French press method, cholic acid method, Ca ²⁺ fusion method, freeze-thaw method, and reverse phase evaporation method. The ethanol dilution method can be carried out, for example, using a microchannel or a vortex.

The cationic lipid may exist in any form, such as lipid nanoparticles (LNP), liposomes, emulsions, micelles, etc. In other words, the lipid membrane structure may exist in any form, such as LNP, liposomes, emulsions, micelles, etc. The term "lipid nanoparticles" (LNP) may mean particles containing lipids as a constituent and having a particle diameter of 100 nm or less. The term "lipid nanoparticles" (LNP) may specifically mean lipid membrane structures having a particle diameter of 100 nm or less. Unless otherwise specified, the term "particle diameter" means the particle diameter measured by dynamic light scattering (DLS). The term "lipid nanoparticles" (LNP) may mean particles containing lipids as a constituent and having an average particle diameter of 100 nm or less. The term "lipid nanoparticle" (LNP) may specifically mean a lipid membrane structure having an average particle size of 100 nm or less. In other words, the lipid membrane structure may have an average particle size of 100 nm or less. Unless otherwise specified, the term "average particle size" means the number-average particle size measured by dynamic light scattering (DLS). Measurement of particle size or average particle size by dynamic light scattering can be performed using a commercially available DLS device such as Zetasizer nano ZS (Malvern).

The antigen contained in the vaccine described herein (hereinafter, simply referred to as "antigen") is not particularly limited. An "antigen" may mean a substance that induces the production of an antibody in an animal body and specifically reacts with the antibody. However, in this specification, nucleic acid is not included in the term "antigen". The antigen can be appropriately selected according to various conditions such as the use of the vaccine described herein. Examples of the antigen include pathogens and components derived therefrom. Examples of pathogens include viruses, bacteria, fungi, parasitic microorganisms, and mycoplasma. The pathogens exemplified above may be, for example, pathogens that cause infectious diseases to which the vaccine described herein is applied. That is, examples of the viruses, bacteria, fungi, parasitic microorganisms, and mycoplasma include viruses, bacteria, fungi, parasitic microorganisms, and mycoplasma that cause infectious diseases to which the vaccine described herein is applied, respectively. The infectious diseases to which the vaccine described herein is applied and the pathogens that cause them will be described later. Examples of components derived from pathogens include components of pathogens and products of pathogens. Specific examples of antigens include inactivated antigens, attenuated antigens, subunit antigens, and recombinant antigens. Inactivated antigens include inactivated viruses, inactivated bacteria, and inactivated toxins (also called "toxoids"). Specific examples of viruses (which may be inactivated before use) include enveloped viruses. An example of an enveloped virus is bovine viral diarrhea virus. Specific examples of bacteria (which may be inactivated before use) include gram-negative bacteria. An example of gram-negative bacteria is Escherichia coli. Another example of gram-negative bacteria is Avibacterium paragallinarum (Haemophilus paragallinarum), which is known as the causative bacterium of infectious coryza, which will be described later. Examples of toxoids include inactivated toxins produced by Clostridium bacteria. Examples of bacteria belonging to the genus Clostridium include Clostridium septicum. Antigens, in terms of chemical structure, include sugars, lipids, peptides, proteins, and chemical or recombinant conjugates thereof (glycolipids, glycoproteins, lipoproteins, etc.). One type of antigen may be used, or two or more types of antigens may be used in combination.

The inactivated antigen can be prepared, for example, by subjecting the target (e.g., a virus, a bacterium, or a toxin) that is the raw material to an inactivation treatment. The inactivation treatment is not particularly limited as long as it does not impair the immunogenicity of the target (in other words, does not impair the immunogenicity of the vaccine described in this specification) and inactivates the target. Examples of inactivation of the target include loss of proliferation and loss of toxicity. The inactivation treatment can be appropriately selected depending on various conditions such as the type of the target. Examples of inactivation treatment include treatment with a detoxifying agent, heat treatment, and ultraviolet irradiation. In particular, examples of inactivation treatment include treatment with a detoxifying agent. As the inactivation treatment, one type of treatment may be used, or two or more types of treatments may be used in combination. Examples of detoxifying agents include formalin, beta-propiolactone (BPL), and binary ethyleneimine (BEI). As the detoxifying agent, one type of component may be used, or two or more types of components may be used in combination.

The vaccines described herein may or may not consist of cationic lipids and antigens. That is, the vaccines described herein may contain other components in addition to cationic lipids and antigens. Examples of the other components include the other components exemplified as components of the lipid membrane structure. Examples of the other components include detoxifying agents. The vaccines described herein may contain other components exemplified as components of the lipid membrane structure, for example, by containing a lipid membrane structure containing other components exemplified as components of the lipid membrane structure. The vaccines described herein may contain a detoxifying agent, for example, by containing an antigen (e.g., a detoxified antigen) containing a detoxifying agent. The other components may be one type of component, or two or more types of components.

The vaccine described herein can be produced, for example, by appropriately mixing the raw materials (i.e., cationic lipid, antigen, and optionally other components). That is, the present specification discloses a method for producing the vaccine described herein, which includes mixing cationic lipid and antigen. Both the cationic lipid and the antigen may or may not be mixed with additional components before mixing. That is, both the cationic lipid and the antigen in the expression "mixing cationic lipid and antigen" may be mixed with other components before mixing. For example, the cationic lipid may be mixed with other components before forming a lipid membrane structure. In addition, after mixing the cationic lipid and the antigen, other components may be further mixed. When the antigen is an inactivated antigen, for example, a previously prepared inactivated antigen may be mixed with the cationic lipid, etc., or an inactivation treatment may be performed after mixing the target (e.g., a virus, a bacterium, or a toxin) that is the raw material of the inactivated antigen with the cationic lipid, etc. When the antigen is an inactivated antigen, preferably, a previously prepared inactivated antigen may be mixed with the cationic lipid, etc.

When the cationic lipid constitutes the lipid membrane structure, the antigen may or may not be encapsulated within the lipid membrane structure. A lipid membrane structure encapsulating an antigen can be produced, for example, by producing a lipid membrane structure in the coexistence of the antigen.

In addition, by mixing a cationic lipid with an antigen, the function of the vaccine can be improved, that is, a vaccine with improved function can be produced. That is, the present specification discloses a method for improving the function of a vaccine, which includes mixing a cationic lipid and an antigen. In addition, the present specification discloses a method for producing a vaccine with improved function, which includes mixing a cationic lipid and an antigen. In other words, the vaccine described in the present specification may be a vaccine with improved function. Specifically, by mixing a cationic lipid with an antigen, the function of the vaccine can be improved compared to a vaccine that does not contain a cationic lipid. Examples of improved vaccine function include enhancement of the immunogenicity of the vaccine (specifically, the immunogenicity of the antigen contained in the vaccine), acceleration of the immune response to the vaccine (specifically, the immune response to the antigen contained in the vaccine), extension of the immune response to the vaccine (specifically, the immune response to the antigen contained in the vaccine), and switching to an immune response different from the immune response induced by the antigen alone contained in the vaccine (for example, switching from a Th1 immune response to a Th2 immune response or switching from humoral immunity to cellular immunity). Improvements in vaccine function, such as enhanced immunogenicity of the antigen contained in the vaccine, can be confirmed, for example, using as an indicator an enhanced immune response to the vaccine (specifically, the immune response to the antigen contained in the vaccine) when the vaccine is administered to an animal. Enhancement of the immune response can be measured, for example, using as an indicator an increase in antibody production against the antigen contained in the vaccine. Examples of increased antibody production include an increase in the amount of antibody production, an increase in the rate of antibody production, and an extension of the period of antibody production. A switch in immune response can be confirmed, for example, by confirming the type of immune response induced.

The content and content ratio of each component (i.e., cationic lipid, antigen, and optionally other components) in the vaccine described herein is not particularly limited as long as the effect of the vaccine described herein is obtained. The content and content ratio of each component (i.e., cationic lipid, antigen, and optionally other components) in the vaccine described herein can be appropriately set according to various conditions such as the type of each component and the use of the vaccine described herein.

The content of the cationic lipid in the vaccine described herein may be, for example, an amount capable of improving the function of the vaccine. The content of the cationic lipid in the vaccine described herein may be, for example, 0.1 μmol/mL or more, 0.2 μmol/mL or more, 0.5 μmol/mL or more, 1 μmol/mL or more, 2 μmol/mL or more, 5 μmol/mL or more, 10 μmol/mL or more, 20 μmol/mL or more, 50 μmol/mL or more, or 100 μmol/mL or more, or 200 μmol/mL or less, 100 μmol/mL or less, 50 μmol/mL or less, 20 μmol/mL or less, 10 μmol/mL or less, 5 μmol/mL or less, 2 μmol/mL or less, 1 μmol/mL or less, 0.5 μmol/mL or less, or 0.2 μmol/mL or less, or a compatible combination thereof. The content of the cationic lipid in the vaccine described herein may be, for example, 0.1-0.2 μmol/mL, 0.2-0.5 μmol/mL, 0.5-1 μmol/mL, 1-2 μmol/mL, 2-5 μmol/mL, 5-10 μmol/mL, 10-20 μmol/mL, 20-50 μmol/mL, 50-100 μmol/mL, or 100-200 μmol/mL. The content of the cationic lipid in the vaccine described herein may be, for example, 0.1-200 μmol/mL, 0.5-50 μmol/mL, or 2-10 μmol/mL. When the vaccine described herein is in a form other than a liquid, "/mL" may be read as "/g".

The content of the antigen in the vaccine described herein may be, for example, an amount that can induce an immune response to the antigen in an animal when the vaccine described herein is administered to the animal.

The shape of the vaccines described herein is not particularly limited. The vaccines described herein may be in any shape, such as a liquid or a powder. The vaccines described herein may be in a liquid form, among others.

The vaccines described herein can be used, for example, by administering to animals. The vaccines described herein can be used, for example, by administering to animals to prevent, reduce, or treat an infectious disease in an animal. That is, the vaccines described herein can be used, for example, to prevent, reduce, or treat an infectious disease in an animal. "Prevention, reduction, or treatment of an infectious disease" also includes prevention, reduction, or treatment of complications caused by the infectious disease, respectively. The infectious disease to which the vaccines described herein are applied may be one type of infectious disease, or two or more types of infectious diseases. The animal to which the vaccines described herein are applied may be one type of animal, or two or more types of animals.

The type of animal is not particularly limited. Examples of animals include livestock animals and pet animals. Examples of livestock animals include cows, pigs, sheep, goats, deer, buffalo, rabbits, horses, chickens, ducks, turkeys, guinea chickens, quails, pheasants, and ostriches. Examples of livestock animals include cows, pigs, and chickens. Examples of pet animals include dogs and cats.

There are no particular restrictions on the type of infectious disease.

Infectious diseases include the following. The "pathogens" listed in parentheses are examples of pathogens that can cause each infectious disease.
Infectious bovine rhinotracheitis (pathogen: infectious bovine rhinotracheitis virus)
Bovine viral diarrhea-mucosal disease (Pathogen: Bovine viral diarrhea virus)
Parainfluenza in cattle (Pathogen: Bovine parainfluenza virus 3)
Bovine respiratory syncytial virus infection (Pathogen: Bovine respiratory syncytial virus)
Bovine adenovirus infection (pathogens: bovine adenoviruses A to C, human adenovirus C, and ovine adenovirus A)
Histophilus somni infection (pathogen: Histophilus somni)
Bovine pasteurellosis (pathogens: Pasteurella multocida, Mannheimia haemolytica, and Bibersteinia trehalosi (Pasteurella trehalosi))
bovine ephemeral fever (pathogen: bovine epidemic virus)
Ibaraki disease (pathogen: Ibaraki virus)
Bovine rotavirus disease (pathogens: rotaviruses A to C)
Bovine coronavirus disease (pathogen: bovine coronavirus)
Bovine colibacillosis (pathogen: Escherichia coli)
Akabane disease (pathogen: Akabane virus)
Chuzan disease (pathogen: Chuzan virus)
Aino virus infection (Pathogen: Aino virus)
Peeton virus infection (pathogen: Peeton virus)
Emphysematous blackleg (pathogen: Clostridium chauvoei)
Malignant edema (pathogen: Clostridium septicum)
Bovine Clostridium perfringens infection (formerly known as bovine necrotic enteritis) (pathogen: Clostridium perfringens)
Enzootic encephalitis (pathogens: Japanese encephalitis virus, West Nile virus, Eastern Equine Encephalitis (EEE) virus, Western Equine Encephalitis (WEE) virus, and Venezuelan Equine Encephalitis (VEE) virus)
Porcine parvovirus infection (pathogen: parvovirus) Porcine Getah virus infection (pathogen: Getah virus)
swine erysipelas (pathogen: Erysipelothrix rhusiopathiae)
Actinobacillus pleuropneumoniae infection in swine (pathogen: Actinobacillus pleuropneumoniae types 1, 2, and 5)
Swine flu (pathogen: influenza A virus)
Infectious bursal disease (pathogen: infectious bursal disease virus)
Newcastle disease (pathogen: Newcastle disease virus)
Infectious bronchitis (pathogen: gamma coronavirus)
Infectious laryngotracheitis (pathogen: Irtovirus)
Infectious coryza (cause: Avibacterium paragallinarum (Haemophilus paragallinarum))
Mycoplasma gallisepticum infection (pathogen: Mycoplasma gallisepticum)
Egg Drop Syndrome-1976 (EDS-76) (Pathogen: Egg Drop Syndrome Virus)
Chicken colibacillosis (pathogen: Escherichia coli)

Infectious diseases also include livestock infectious diseases (legal infectious diseases) and notifiable infectious diseases as defined in the Livestock Infectious Diseases Control Act. Livestock infectious diseases (legal infectious diseases) and notifiable infectious diseases as defined in the Livestock Infectious Diseases Control Act, as well as the pathogens that may cause them, can be found, for example, on the NARO website (https://www.naro.affrc.go.jp/org/niah/disease_fact/kansi.html).

The method of administration of the vaccine described herein is not particularly limited as long as the effect of the vaccine described herein can be obtained. The method of administration of the vaccine described herein can be appropriately selected according to various conditions such as the type of animal, the type of infectious disease, and the type of antigen. The administration route of the vaccine described herein includes intramuscular administration, intradermal administration, subcutaneous administration, intraocular administration, intratracheal administration, and intranasal administration. The administration route of the vaccine described herein includes, in particular, intramuscular administration, intradermal administration, subcutaneous administration, and intraocular administration. In one aspect, the vaccine described herein is expected to be able to suppress, for example, side reactions at the inoculation site, and therefore the vaccine described herein may be suitable for administration routes such as intramuscular administration, intradermal administration, subcutaneous administration, and intraocular administration. The vaccine described herein can be administered to animals as it is, or after being appropriately prepared into a form suitable for administration. For example, the vaccine described herein, which is a liquid, may be administered to animals as it is, or after being appropriately diluted, etc. Also, for example, the vaccine described herein, which is not a liquid, may be administered to animals as it is, or after being appropriately prepared into a liquid.

The vaccines described herein may be used alone or as a mixed vaccine formulation with other vaccines. That is, the present invention also discloses a mixed vaccine formulation of the vaccines described herein and other vaccines. The other vaccines include vaccines for preventing, reducing, or treating infectious diseases in animals. The infectious disease to which the other vaccine is applied may or may not be the same as the infectious disease to which the vaccine described herein is applied. The infectious disease to which the other vaccine is applied may be one type of infectious disease, or may be two or more types of infectious diseases. The animal to which the other vaccine is applied may or may not be the same as the animal to which the vaccine described herein is applied. The other vaccine may typically be applied to at least some or all of the animals to which the vaccine described herein is applied. The animal to which the other vaccine is applied may be one type of animal, or may be two or more types of animals. As the other vaccine, one type of vaccine may be used, or two or more types of vaccines may be used.

The dosage of the vaccine described herein is, in terms of the dosage of cationic lipid per dose, for example, 1 nmol or more, 2 nmol or more, 5 nmol or more, 10 nmol or more, 20 nmol or more, 50 nmol or more,
The amount of the vaccine may be 1 nmol or more, 100 nmol or more, 200 nmol or more, or 500 nmol or more, or 1000 nmol or less, 500 nmol or less, 200 nmol or less, 100 nmol or less, 50 nmol or less, 20 nmol or less, 10 nmol or less, 5 nmol or less, or 2 nmol or less, or any combination thereof that is not inconsistent. The dosage of the vaccine described herein may be, for example, 1-2 nmol, 2-5 nmol, 5-10 nmol, 10-20 nmol, 20-50 nmol, 50-100 nmol, 100-200 nmol, 200-500 nmol, or 500-1000 nmol, in terms of the dosage of cationic lipid per dose. The dosage of the vaccines described herein may be, specifically, for example, 1 to 1000 nmol, 2 to 500 nmol, or 5 to 200 nmol, calculated as the dosage of cationic lipid per dose.

The vaccines described herein may be administered, for example, once, twice or more.

<2> Use of the vaccine The vaccine described herein can be used, for example, to prevent, reduce, or treat an infectious disease in an animal. That is, the present specification provides a method for preventing, reduce, or treat an infectious disease in an animal, the method comprising a step of administering the vaccine described herein to an animal.

The animals and infectious diseases to which the vaccines described in this specification are applicable are as described above.

The administration route, dosage, and other administration methods for the vaccines described in this specification are as described above.

The present invention will now be described in more detail with reference to the following non-limiting examples.

Example 1 Preparation of Adjuvant and Evaluation of Physical Properties 1. Preparation of Adjuvant ssPalmEC-LNP was used as an adjuvant in Examples 1 to 3. ssPalmEC-LNP was prepared by the following procedure.

(1) Preparation of ethanol solutions of each lipid
ssPalmE-P4C2 (COATSOME® SS-EC, Yuka Sangyo Co., Ltd.), cholesterol (Sigma), and DOPE (18:1 deruta9-cis 1,2-dioleolyl-sn-glycero-3-phosphoethanolamine, Avanti) were each dissolved in 99.5% ethanol to prepare a 5 mM ethanol solution. DMG-PEG 2000 (SUN BRIGHT®, Yuka Sangyo Co., Ltd.) was dissolved in 99.5% ethanol to prepare a 1 mM ethanol solution. Each lipid ethanol solution was stored at -20°C.

(2) Preparation of Buffer Solution Malic acid (Nacalai Tesque, Inc.) and sodium chloride (Nacalai Tesque, Inc.) were dissolved in distilled water and adjusted to pH 3.0 with NaOH to prepare a 20 mM malic acid/30 mM sodium chloride aqueous solution (pH 3.0) (hereinafter also referred to as "malic acid buffer solution"). The malic acid buffer solution was filtered through a filter with a pore size of 0.2 μm and stored at 4° C.

MES (2-(N-morpholino)ethanesulfonic acid, Nacalai Tesque, Inc.) was dissolved in distilled water and adjusted to pH 5.5 with NaOH to prepare a 20 mM MES aqueous solution (pH 5.5) (hereinafter also referred to as "MES acid buffer"). The MES buffer was filtered through a filter with a pore size of 0.2 μm and stored at 4°C.

(3) Preparation of ssPalmEC-LNPs by ethanol dilution method
0.473 mL of 5 mM ssPalmE-P4C2 ethanol solution, 0.237 mL of 5 mM DOPE ethanol solution, 0.079 mL of 5 mM cholesterol ethanol solution, and 0.118 mL of 1 mM DMG-PEG 2000 ethanol solution (molar ratio of ssPalmE-P4C2:DOPE:cholesterol:DMG-PEG 2000 = 60:30:10:3) were mixed, and 3.093 mL of 99.5% ethanol was added to dilute and prepare 4 mL of lipid ethanol mixed solution. 0.4 mL of lipid ethanol mixed solution and 0.6 mL of malic acid buffer were mixed for 80 seconds using a microchannel (iLiNP (registered trademark) 1.0, Lilac Pharma) at 0.3 mL/min of lipid ethanol mixed solution, 0.45 mL/min of malic acid buffer, and a total of 0.75 mL/min. This was repeated 10 times to obtain 10 mL of ssPalmEC-LNP solution. After adding an equal amount of MES acid buffer and mixing, the buffer and ethanol were replaced with D-PBS(-) (phosphate buffered saline, manufactured by Nacalai Tesque, Inc.) using an ultrafiltration membrane. Specifically, the ssPalmEC-LNP solution diluted with MES buffer was placed in the cup of an Amicon Ultra-15 tube (molecular weight cutoff 50 kDa, manufactured by Sigma) and centrifuged at 1000 × g to concentrate. D-PBS(-) was added to the concentrated solution up to the mark and centrifuged again under the same conditions. This operation was repeated once more to recover the concentrated solution. The remaining ssPalmEC-LNP was recovered while washing the ultrafiltration membrane with D-PBS(-) to make the total volume 1 mL. The recovered ssPalmEC-LNP was stored at 4 ° C. The lipid concentration of the recovered ssPalmEC-LNP was 4 μmol/mL.

2. Evaluation of the physical properties of adjuvants
The particle size, polydispersity index (PdI), and zeta potential of ssPalmEC-LNP were measured by dynamic light scattering (Zetasizer nano ZS, Malvern). The measurement results are shown in Table 1.

Example 2 Vaccine Preparation and Evaluation (1)
1. Vaccine preparation (1) Preparation of inactivated bovine viral diarrhea virus type 1 (Nose-KB strain)
MDBK cells (obtained from the RIKEN cell bank and adapted to suspension culture) were cultured in suspension at 37°C in a cell growth medium (Eagle's MEM (Nissui Pharmaceutical Co., Ltd.) containing 3% tryptose phosphate broth and 0.03% L-glutamine) supplemented with 3 vol% fetal bovine serum (Hyclone). The cell concentration was adjusted to 8.6 x ¹⁰⁵ cells/mL, and the Nose-KB strain of bovine viral diarrhea virus type 1 was inoculated to a concentration of ^104.0 TCID50 or _more per mL. The cell and virus suspension was inoculated into a virus propagation medium (Eagle MEM (Nihon Seiyaku) containing 2.5 vol% lactalbumin hydrolysate solution, 0.1 w/v% bactopeptone, and 0.15 w/v% glucose) containing 1 vol% fetal bovine serum (Low IgG) (Invitrogen) and rotated at 37°C for 3 days, then centrifuged at 12,800 x g to collect the supernatant. The culture supernatant after centrifugation was concentrated to about 1/10 of its original volume using an ultrafiltration membrane with a molecular weight cutoff of 50 kDa (KrosFlo, K25S-300-01N). Formalin (Ken-ei Pharmaceutical) was added to the resulting virus suspension to a concentration of 0.1 vol%. The virus was inactivated by sensitization at 2 to 5°C for 4 weeks. This was used as the immunization antigen BVD1-NoK.

(2) Vaccine preparation ssPalmEC-LNP was added to BVD1-NoK antigen with a pre-inactivated virus amount of 10 ^8.72 TCID ₅₀ at 50 nmol, 150 nmol, and 450 nmol (as total lipid amount) per dose, respectively, and the mixture was adjusted to 12 mL with phosphate buffer and Eagle's MEM containing 0.1 vol% formalin to prepare experimental vaccines A, B, and C, respectively. In addition, experimental vaccine D was prepared by adjusting the same amount of BVD1-NoK antigen as experimental vaccines A to C to 12 mL with phosphate buffer and Eagle's MEM containing 0.1 vol% formalin.

2. Safety Evaluation The vaccines A to D were each inoculated into rats as follows, and the safety of ssPalmEC-LNP as an adjuvant was evaluated.

Twenty-two rats weighing approximately 100 g were used, and 5 rats each were given 1.0 mL of the test vaccines A to D by intramuscular injection into the left and right thighs to form groups A to D, while the remaining 2 rats served as a control group (group E) that was not vaccinated. Each rat was kept for 21 days. As a result, no abnormalities were observed in clinical symptoms after vaccination or local reactions at the vaccination site 21 days after vaccination in any of groups A to D. These results demonstrated that ssPalmEC-LNP is highly safe.

3. Efficacy evaluation In the above safety evaluation, blood was collected from rats on the 21st day after vaccination to obtain serum. The serum (a total of 22 samples from 4 groups x 5 rats + 2 rats in the control group) was used to measure the neutralizing antibody titer. The neutralizing antibody titer was measured based on 3.5.7.2.2 of the Animal Biological Products Standards "Infectious bovine rhinotracheitis, bovine viral diarrhea bivalent, bovine parainfluenza, bovine respiratory syncytial virus infection mixed (adjuvanted) inactivated vaccine" (Ministry of Agriculture, Forestry and Fisheries Notification No. 1567, October 3, 2002). MDBK-NST cells (purchased from the RIKEN cell bank) were used as cells. The highest dilution ratio of serum that inhibited CPE on two wells of cultured cells was defined as the neutralizing antibody titer. The geometric mean (GM) of the neutralizing antibody titer of each group was calculated and a t-test was performed.

The results are shown in Figure 1. The antibody titer did not increase in Group D (vaccinated without ssPalmEC-LNP) and Group E (non-vaccinated), but increased in the vaccinated groups (Groups A to C) with ssPalmEC-LNP. In addition, the t-test showed that the antibody titer in Group B was significantly higher than that in Group D. These results demonstrated that ssPalmEC-LNP has an effect as an adjuvant. Furthermore, the results in Group B exceeded the standard value of the Animal Biological Products Standards "Inactivated Vaccine for Infectious Bovine Rhinotracheitis, Bovine Viral Diarrhea, Bovine Parainfluenza, and Bovine Respiratory Syncytial Virus Infection (with Adjuvant)" (Ministry of Agriculture, Forestry and Fisheries Notification No. 1567, October 3, 2002), and the results passed the national examination.

Example 3 Vaccine Preparation and Evaluation (2)
1. Preparation of vaccine Toxoid of Clostridium septicum was used as an antigen. C. septicum No. 44T strain was cultured for 20 hours (at 37°C) in cooked meat medium (Becton Dickinson and Company), and then subcultured in porcine BHI medium (Becton Dickinson and Company) containing 0.3 w/v% glucose (Fujifilm Wako Pure Chemical Industries, Ltd.) and 0.05 w/v% L-cysteine hydrochloride monohydrate (Nacalai Tesque, Inc.) for 24 hours. The culture medium was centrifuged (at 12,800 x g) to collect the culture supernatant. The toxin in the culture supernatant was measured using the 3.1.2 toxicity test method of Bovine Clostridium Infection 5-Type Mixed (Adjuvant Added) Toxoid (Ministry of Agriculture, Forestry and Fisheries Notification No. 646, April 22, 2010) of the Animal Biological Products Standards (Ministry of Agriculture, Forestry and Fisheries Notification No. 646) and was found to be 12,800 CU/mL. The culture supernatant was detoxified by adding 0.4 vol% formalin (Ken-ei Pharmaceutical Co., Ltd.) and treating at 37°C for 3 days. The treated product was concentrated 26-fold, added with 0.1 vol% formalin, and treated at 37°C overnight to obtain the toxoid to be used as an antigen. ssPalmEC-LNP was added to the toxoid equivalent to 57,574 CU per dose in the amounts shown in Table 1, and phosphate buffered saline (PBS) was added to make the total volume 0.4 mL to obtain the experimental vaccines A to D.

2. Potency test 0.4 mL of each of the vaccines A to D was administered intramuscularly to the thigh of guinea pigs (Hartley, 5 weeks old, female) (first vaccination). Two weeks after the first vaccination, the same amount of the vaccine was administered (second vaccination), and blood was collected from the heart under appropriate anesthesia on the 10th day after the second vaccination. The blood was centrifuged at 3500 rpm for 15 minutes to separate the serum. The serum neutralizing antibody titer was measured according to the following procedure based on the Standards for Animal Biological Products Bovine Clostridium Infection 5-Type Mixed (Adjuvanted) Toxoid (Ministry of Agriculture, Forestry and Fisheries Notification No. 646, April 22, 2010).

The serum was diluted 2-fold from 5-fold with cell culture medium (Eagle MEM (Nihon Pharmaceutical) supplemented with 0.295 w/v% tryptose phosphate broth, 0.0292 w/v% L-glutamine, and 5 v/v% fetal bovine serum (inactivated at 56°C for 30 minutes)). The toxin used for neutralization was C. septicum culture supernatant filtered through a membrane filter (Millipore) with a pore size of 450 nm or less and diluted with cell culture medium to make 4 CU. The diluted serum and the toxin were mixed in equal amounts and neutralized at 37°C for 1 hour to prepare the sample. 100 μL of the sample was added to a cell sheet of Vero cells in a 96-well plate, and the cells were cultured at 37°C under 5 vol% carbon dioxide for 1 day, and the cytopathic effect (CPE) was observed. The highest dilution of serum that suppressed CPE in more than 50% of cultured cells was defined as the neutralizing antibody titer.

The results are shown in Table 2. The induced neutralizing antibody titer increased depending on the amount of ssPalmEC-LNP mixed. Compared to the group administered antigen only (vaccine D group), the 50 nmol lipid/d group (vaccine B group) showed approximately 8 times the neutralizing antibody titer, and the 150 nmol lipid/d group (group C) showed approximately 18 times the neutralizing antibody titer, demonstrating that ssPalmEC-LNP has an effect as an adjuvant. Furthermore, the values of groups B and C were above the standard value of the Animal Biological Products Standards Bovine Clostridium Infection 5-Type Mixed (Adjuvanted) Toxoid (Ministry of Agriculture, Forestry and Fisheries Notification No. 646, April 22, 2010), and the results were sufficient to pass the national examination.

3. Safety Confirmation Test The safety confirmation test was conducted simultaneously with the potency test. Each guinea pig was identified by an animal marker. Body weights and clinical observations were performed on the day, 1 day, and 2 days after the first vaccine administration in the potency test.

As a result, no significant changes were observed in local or systemic clinical observations in any of the groups. Body weight measurements showed no significant difference between the weight of the group administered vaccine D and the groups administered vaccines A, B, C on the same day.

Example 4 Preparation of adjuvant (2)
The lipid ethanol solutions used for the preparation of ssPalmEC-LNP, SM-102-LNP, and ALC-0315-LNP were prepared as follows: SM-102 and ALC-0315 are the names of cationic lipids contained in the novel coronavirus vaccines SpikeVax and Comirnaty, respectively.
ssPalmEC-LNP: ssPalmE-P4C2 (COATSOME (registered trademark) SS-EC, NOF Corporation), cholesterol (Tokyo Chemical Industry Co., Ltd.), DOPE (COATSOME ME-8181, NOF Corporation), and DMG-PEG-2000 (SUNBRIGHT GM-020, NOF Corporation) were dissolved in 99.5% ethanol to prepare stock ethanol solutions of 10 mg/mL ssPalmE-P4C2, 1.935 mg/mL cholesterol, 3.72 mg/mL DOPE, and 2.503 mg/mL DMG-PEG-2000 (middle column of Table 3).
The stock ethanol solution was mixed with ethanol in the volume shown in the "LNP preparation" column of Table 3 to prepare lipid ethanol solutions for ssPalmEC-LNP.

SM-102-LNP: SM-102 (Cayman), cholesterol (Tokyo Chemical Industry Co., Ltd.), DSPC (COATSOME MC-8080, NOF Corporation), and DMG-PEG-2000 (SUNBRIGHT GM-020, NOF Corporation) were dissolved in 99.5% ethanol to prepare stock ethanol solutions of 100 mg/mL SM-102, 7.731 mg/mL cholesterol, 15.8 mg/mL DSPC, and 25.03 mg/mL DMG-PEG-2000 (middle column of Table 4).
The stock ethanol solution was mixed with ethanol in the volume shown in the "LNP preparation" column of Table 4 to prepare lipid ethanol solutions for SM-102-LNP.

ALC-0315-LNP: ALC-0315 (Cayman Chemical), cholesterol (Tokyo Chemical Industry Co., Ltd.), DSPC (COATSOME MC-8080, NOF Corporation), and ALC-0159 (Cayman Chemical) were dissolved in 99.5% ethanol to prepare stock ethanol solutions of 50 mg/mL ALC-0315, 7.731 mg/mL cholesterol, 15.8 mg/mL DSPC, and 100 mg/mL ALC-0159 (middle column of Table 5).
The stock ethanol solution was mixed with ethanol in the volume shown in the "LNP preparation" column of Table 5 to prepare lipid ethanol solutions for ALC-0315-LNP.

Each LNP was prepared according to the ethanol dilution method. That is, LNP was prepared using the lipid ethanol solution for each LNP prepared as described above using a microchannel (iLiNP (registered trademark) 1.0, Lilac Pharma Co., Ltd.). Two syringe pumps, YSP-101 (standard type, YAC Co., Ltd.), were used. The lipid ethanol solution and PBS (Nacalai Tesque Co., Ltd.) were filled into glass syringes, respectively, and the lipid ethanol solution for LNP was injected into the microchannel at a flow rate of 0.45 mL/min and PBS at 0.30 mL/min, and mixed to obtain an LNP solution. The prepared LNP solution was buffer-substituted with PBS using ultrafiltration (Amicon Ultra-15 tube, 100 kDa, Merck Co., Ltd.) until the calculated ethanol concentration was less than 1%. The prepared LNP was stored at 4°C.
The physical property data of each LNP is shown in Table 6.

Example 5 Vaccine Preparation and Evaluation (3)
1. Preparation of vaccines The antigen used was a toxoid of Clostridium septicum prepared in the same manner as in Example 3. The toxoid equivalent to 57,574 CU per dose was mixed with ssPalmEC-LNP, SM-102-LNP, and ALC-0315-LNP prepared in Example 4 according to the adjuvant column in Table 7. Since each LNP has a different lipid composition, the amount of cationic lipid was added so that it was 400 nmol per dose. Phosphate buffered saline (PBS) was added to make the total volume 0.4 mL to prepare test vaccines E to G. As a control without adjuvant, test vaccine H was prepared using only PBS and the same amount of antigen as test vaccines E to G (Table 7).

2. Potency test The vaccines E to H were administered to five guinea pigs in the same manner as in Example 3, and the neutralizing antibody titers of the serum were measured. The results are shown in the neutralizing antibody titer column in Table 7. When a Student-t test (significance level <0.05) was performed using the neutralizing antibody titers of each individual, the vaccine E was significantly higher than the vaccine H, but no significant difference was observed between the vaccines F and G. The vaccine E also had a significantly higher neutralizing antibody titer against the vaccines F and G. From the above, it was shown that ssPalmEC-LNP induces a significantly higher neutralizing antibody titer than ALC-0315-LNP and SM-102-LNP when the molar number of cationic lipids was the same. The neutralizing antibody titers of the equivalent pooled serum from each group were higher for the vaccines E and G than for the vaccine H. The neutralizing antibody titers of the vaccine F were the same.

3. Safety Confirmation Test As in Example 3, a safety confirmation test was carried out simultaneously with the potency test.
As a result, no significant changes were observed in local or systemic clinical observations in any of the groups. Body weight measurements showed no significant difference between the weights of the group administered investigational vaccine H and the groups administered investigational vaccines E to G on the same day.

Example 6 Preparation of adjuvant (3)
The lipid ethanol solutions for each LNP used in the preparation of ssPalmEC-LNP, ssPalmOP-LNP, and LNP w/o cationic lipid were prepared as follows (note that w/o means without).
ssPalmEC-LNP: ssPalmE-P4C2 (COATSOME (registered trademark) SS-EC, NOF Corporation), cholesterol (Tokyo Chemical Industry Co., Ltd.), DOPE (COATSOME ME-8181, NOF Corporation), and DMG-PEG-2000 (SUNBRIGHT GM-020, NOF Corporation) were dissolved in 99.5% ethanol to prepare stock ethanol solutions of 7.01 mg/mL ssPalmE-P4C2, 1.935 mg/mL cholesterol, 3.72 mg/mL DOPE, and 2.503 mg/mL DMG-PEG-2000 (middle column of Table 8).
The stock ethanol solution was mixed with ethanol in the volume shown in the "LNP Preparation" column of Table 8 to prepare lipid ethanol solutions for ssPalmEC-LNP.

ssPalmOP-LNP: ssPalmOP (COATSOMESS-OP, NOF Corp.), cholesterol (Tokyo Chemical Industry Co., Ltd.), DOPE (COATSOME ME-8181, NOF Corp.), and DMG-PEG-2000 (SUNBRIGHT GM-020, NOF Corp.) were dissolved in 99.5% ethanol to prepare stock ethanol solutions of 10 mg/mL ssPalmOP, 1.935 mg/mL cholesterol, 3.72 mg/mL DOPE, and 2.503 mg/mL DMG-PEG-2000 (middle column of Table 9).
The stock ethanol solution was mixed with ethanol in the volume shown in the "LNP preparation" column of Table 9 to prepare lipid ethanol solutions for ssPalmOP-LNP.

LNP w/o cationic lipid: Cholesterol (Tokyo Chemical Industry Co., Ltd.), DOPE (COATSOME ME-8181, NOF Corp.), and DMG-PEG-2000 (SUNBRIGHT GM-020, NOF Corp.) were dissolved in 99.5% ethanol to prepare stock ethanol solutions of 1.935 mg/mL cholesterol, 4 mg/mL DOPE, and 2.503 mg/mL DMG-PEG-2000 (middle column of Table 10).
The stock ethanol solution was mixed with ethanol in the volume shown in the "LNP preparation" column of Table 10 to prepare lipid ethanol solutions for LNP w/o cationic lipid.

Each LNP was prepared according to the ethanol dilution method as in Example 4. That is, LNP was prepared using the lipid ethanol solution for each LNP prepared as described above using a microchannel (iLiNP (registered trademark) 1.0, Lilac Pharma). The LNP solution was obtained at a flow rate of 0.36 mL/min for the lipid ethanol solution for LNP and 0.12 mL/min for PBS, for a total of 0.48 mL/min. The prepared LNP solution was replaced with 0.01 mol/L Tris-HCl 10% sucrose buffer (pH 7.5) using an ultrafiltration membrane. The prepared LNP was stored at 4°C. The physical property data at this time are shown in Table 11.

Example 7 Vaccine Preparation and Evaluation (4)
1. Preparation of vaccines As an antigen, a toxoid of Clostridium septicum obtained in the same manner as in Example 3 was used. The toxoid equivalent to 57,574 CU per dose was mixed with ssPalmEC-LNP and ssPalmOP-LNP prepared in Example 4 according to the adjuvant column in Table 12. Since the lipid composition of the two LNPs is different, the total lipid amount was added to 800 μg per dose. Phosphate buffered saline (PBS) was added to make the total volume 0.4 mL to prepare the experimental vaccines I to K. As a control, experimental vaccine L without the addition of adjuvant was set (Table 12).

2. Potency test Test vaccines I to L were inoculated into guinea pigs in the same manner as in Example 3, and serum neutralizing antibody titers were measured. A Student-t test was performed at a significance level of 5%, and it was shown that ssPalmEC-LNP (test vaccine I) induced a significantly higher neutralizing antibody titer than ssPalmOP-LNP (test vaccine J) and LNP w/o cationic lipid (test vaccine K) (Table 12). The neutralizing antibody titers of equal amounts of pooled serum from each group were slightly higher for test vaccines J and K than for test vaccine L. Test vaccine J was also slightly higher than test vaccine K, suggesting a weak adjuvant effect, although the difference was not significant.

3. Safety Confirmation Test A safety confirmation test was carried out in the same manner as in Example 3. As a result, no significant changes were observed in local and systemic clinical observations in any of the groups. In addition, when a Student-t test was carried out for each group at a significance level of 5%, no significant difference was observed between the weight gain ratio of the non-adjuvanted control (investigative vaccine L) administration group and the experimental vaccines J and K administration groups. In the experimental vaccine I administration group, the weight gain ratio on the first day after the first vaccination was significantly lower than that of the experimental vaccine L administration group, but was equivalent to that of the experimental vaccine L administration group on the second day after the first vaccination.

Example 8 Preparation of adjuvant (4)
The lipid ethanol solutions used in the preparation of ssPalmEC-LNP were prepared as described in Example 6.
The preparation of ssPalmEC-LNP was performed according to the ethanol dilution method as in Example 6. That is, the lipid solutions were mixed in the amounts shown in Table 13 to prepare lipid ethanol solutions. A microchannel was used to prepare LNP. The lipid ethanol solution was mixed at a flow rate of 0.36 mL/min, PBS at 0.12 mL/min, and a total of 0.48 mL/min until the lipid ethanol solution was used up. The prepared LNP solution was buffer-substituted with 0.01 mol/L Tris-HCl in 10% sucrose buffer (pH 7.5) using an ultrafiltration membrane. The prepared LNP was stored at 4°C.
The physical property data of this LNP is shown in Table 14.

Example 9 Vaccine Preparation and Evaluation (5)
1. Preparation of inactivated bacteria In accordance with the Animal Biological Products Standards, Newcastle Disease, Infectious Bronchitis, Infectious Coryza (Types A and C), and Mycoplasma Gallisepticum Infection Mixed (Oil-Based Adjuvant-Added) Inactivated Vaccine (Seed) (Ministry of Agriculture, Forestry and Fisheries Notification No. 134, January 27, 2023), the antigen was prepared as follows: Avibacterium paragallinarum type C KA strain was inoculated into 3.7 w/v% porcine-derived BHI medium containing 5 v/v% inactivated chicken serum that was negative for antibodies against infectious coryza types A and C, and 0.01 w/v% β-NAD, and cultured at 37°C for 24 hours. The cultured bacterial liquid was treated with a surfactant, then concentrated by centrifugation, and 0.1 vol% β-propiolactone was added. The cultured bacterial liquid was inactivated by leaving it at 2 to 5°C for 44 hours and used as an antigen in the test described below.

2. Vaccine preparation The number of live bacteria before inactivation was 3.6 x ¹⁰⁷ per dose, and ssPalm EC-LNP prepared in Example 6 was added as an adjuvant at 50 nmol, 200 nmol, and 800 nmol per dose, respectively, and adjusted with phosphate buffered saline (PBS) to give experimental vaccines A, B, and C. In addition, experimental vaccine D was prepared by adding only the same amount of antigen as experimental vaccines A to C in PBS without using an adjuvant (Table 15).

3. Vaccination and evaluation Ten SPF chickens were used for each test group. Four-week-old SPF chickens were immunized with the four vaccines prepared by injecting 0.5 mL of each immunogen into the leg muscle, and then raised for five weeks. Clinical observations were also conducted throughout the test period, and the inoculation site was observed.
Five weeks after immunization, blood samples were taken to obtain serum. Antibody titers (reaction inhibition rates) against the antigen were measured using enzyme immunoassay (competitive ELISA) according to the following procedure. All samples were measured in duplicate, and the average value was calculated for each test group. The significance of the difference from the control group was tested using Student's t-test.

The antigen (inactivated Avibacterium paragallinarum type C KA strain) was sonicated and diluted to 5 μg/mL in 50 mM carbonate/bicarbonate buffer, and 100 μL was added to each well of a 96-well ELISA plate (Fisher Scientific) and coated overnight. The plate was washed with PBS containing 0.05% Tween (PBS-T) and blocked with PBS-T containing 1% bovine serum albumin (BSA) (Wako) at 30°C for 1 hour. After washing the plate with PBS-T, 100 μL of each serum sample, reference negative serum, and reference positive serum were added to 2 wells (duplicate measurement) and incubated at 30°C for 1 hour. The plate was washed with PBS-T, and 100 μL/well of anti-IC-type C monoclonal antibody (6 ng/mL) was added and sensitized at 30°C for 1 hour. The plate was washed with PBS-T, and 100 μL/well of HRP-labeled anti-mouse IgG antibody was added at a 16,000-fold dilution for 1 hour at 30°C. After washing with PBS-T, 100 μl/well of TMB substrate kit (Thermo Fisher) was added and incubated for 30 minutes at 30°C. The reaction was stopped by adding 100 μl/well of 1M sulfuric acid. The absorbance at 450 nm was measured, and the reaction inhibition rate was calculated using the following formula, which was used as the titer.
Reaction inhibition rate (%) = 100 - [(OD of test serum x 100) ÷ (OD of negative serum)]
Statistical differences between titers were tested using the Student's t-test at a significance level of 1%.

4. Safety evaluation No abnormalities were observed in the chickens due to vaccination throughout the test period. In addition, no side effects such as swelling or induration were observed at the injection site after immunization.

5. Evaluation of antibody induction ability
Groups A to C, which received the vaccine using ssPalm EC-LNP as an adjuvant, showed a statistically significant increase in antibody titers compared to group D, which did not use an adjuvant.
There was a positive correlation with the concentration of EC-LNP (Table 16, FIG. 2). These results demonstrated that ssPalm EC-LNP has a dose-dependent adjuvant effect in chickens.

Claims (23)

A vaccine comprising a cationic lipid and an antigen,
The vaccine, wherein the cationic lipid is a compound represented by the following general formula (I):

[Wherein,
Each of na and nb independently represents an integer of 1 to 4.
^Xa and ^Xb each independently represent the following structure ^X1 or the following structure ^X2 :
^R7 represents an alkyl group having 1 to 6 carbon atoms;
p represents an integer of 1 to 4;

R ^1a and R ^1b each independently represent R ³ or R ⁴ ;
^R3 represents -( _CH2 ) _n1- ;
^R4 represents _-C6H4- ( _CH2 ) _n3 - _COO- ( _CH2 ) _n2- ;
n1 represents an integer of 1 to 5;
n2 represents an integer of 1 to 5;
n3 represents an integer from 1 to 3;
^R2a and ^R2b each independently represent ^R5 or ^R6 ;
^R5 represents a functional group obtained by removing a carboxy group from a monoester of succinic acid or glutaric acid with a fat-soluble vitamin having a hydroxyl group,
^R6 represents an aliphatic hydrocarbon group having 12 to 22 carbon atoms. The vaccine according to claim 1, which satisfies any one of the following conditions (1) to (4) and any one of the following conditions (5) to (8):
(1) na is 2, ^Xa is ^X1 , ^R1a is R3 in which n1 is ³ , and ^R2a is ^R5 ;
(2) na is 2, ^Xa is ^X2 , ^R1a is ^R3 with n1 being 2, and ^R2a is ^R5 ;
(3) na is 2, ^Xa is ^X2 , ^R1a is ^R3 with n1 being 2, and ^R2a is ^R6 ;
(4) na is 2, ^Xa is ^X2 , ^R1a is ^R4 , n2 is 2 and n3 is 1, and ^R2a is ^R6 ;
(5) nb is 2, ^Xb is ^X1 , ^R1b is R3 with n1 being ³ , and ^R2b is ^R5 ;
(6) nb is 2, ^Xb is ^X2 , ^R1b is ^R3 with n1 being 2, and ^R2b is ^R5 ;
(7) nb is 2, ^Xb is ^X2 , ^R1b is ^R3 with n1 being 2, and ^R2b is ^R6 ;
(8) nb is 2, ^Xb is ^X2 , ^R1b is ^R4 , n2 is 2 and n3 is 1, and ^R2b is ^R6 . The vaccine of claim 1 or 2, wherein p is 3 or 4. 3. The vaccine of claim 1 or 2, wherein na is identical to nb, ^Xa is identical to ^Xb , ^R1a is identical to ^R1b , and ^R2a is identical to ^R2b . The vaccine of claim 1 or 2, wherein the cationic lipid is a component of ssPalmEC and/or ssPalmE. The vaccine according to claim 1 or 2, wherein the cationic lipid is a constituent lipid of a lipid membrane structure. The vaccine according to claim 6, wherein the lipid membrane structure further contains a non-cationic lipid. The vaccine of claim 7, wherein the non-cationic lipid is one or more components selected from the group consisting of phospholipids, PEG lipids, and steroids. The vaccine of claim 8, wherein the steroid is one or more components selected from the group consisting of cholesterol and its derivatives. The vaccine according to claim 1 or 2, wherein the antigen is one or more components selected from the group consisting of pathogens and components derived therefrom. The vaccine according to claim 1 or 2, wherein the antigen is an inactivated antigen. The vaccine of claim 11, wherein the inactivated antigen is an inactivated virus. The vaccine of claim 11, wherein the inactivated antigen is an inactivated bacterium. The vaccine of claim 11, wherein the inactivated antigen is a toxoid. The vaccine of claim 12, wherein the virus is bovine viral diarrhea virus. The vaccine of claim 13, wherein the bacterium is Avibacterium paragallinarum. The vaccine of claim 14, wherein the toxoid is a toxoid of Clostridium septicum. The vaccine according to claim 1 or 2 for preventing, reducing or treating an infectious disease in an animal. The vaccine of claim 18, wherein the animal is a livestock animal or a pet animal. The vaccine according to claim 1 or 2, which is administered by a route selected from the group consisting of intramuscular administration, intradermal administration, subcutaneous administration, and intraocular administration. The vaccine of claim 1 or 2, which is administered intramuscularly. A method for producing a vaccine, comprising the steps of:
The vaccine is a vaccine according to claim 1 or 2,
mixing said cationic lipid and said antigen. The method of claim 22, wherein the vaccine is an improved vaccine.

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